Cellular DNA can be chemically modified and hence damaged when exposed to environmental agents, metabolic byproducts, or chemotherapeutic agents. The most toxic of these lesions is the interstrand cross-link (ICL), a covalent bridge formed between the complementary strands of the DNA double helix. If not repaired, an ICL provides a physical constraint that prevents DNA strand separation during replication and transcription. The mammalian Mus81-Eme1 complex is required for efficient repair of ICLs as demonstrated by the sensitivity of Mus81 knockout mice, and cells derived from these mice, to ICL inducing agents. Mus81-Eme1 has structure specific nuclease activity and Mus81 shares homology with the XPF/Rad1 family of proteins that can cleave intact and nicked Holliday junctions, replication fork structures, and 3'flaps in vitro. However, it is not known how Mus81-Eme1 functions to repair ICLs in vivo. This proposal aims to address how Mus81-Eme1 functions to maintain genomic stability and aid ICL repair while furthering our understanding of the mammalian repair factors and pathways that coordinate to repair these toxic lesions. These aims will be addressed by a combination of genetic, biochemical and cell based approaches. In order to determine which pathway Mus81-Eme1 acts in, RNAi will be used to target known ICL repair factors in Mus81 wild type and deficient cells. Damage sensitivity assays will be performed on the resulting stable cell lines. In addition, factors involved in replication associated ICL repair will be isolated and identified using a DNA affinity purification system coupled with MudPIT proteomics. The contribution of known and newly identified factors to ICL repair will be determined by replication coupled in vivo ICL repair assays, and immunofluorescence studies. Understanding how Mus81 contributes to genomic stability by functioning in ICL repair will add to our knowledge of the cellular mechanisms that act to eliminate these critical DNA lesions. In addition, identification and mechanistic understanding of proteins mediating ICL repair reactions will enable development of more effective cancer therapeutic agents and strategies. Relevance to public health: DNA interstrand cross-links are a form of toxic DNA damage which is generated by a class of compounds used extensively in cancer chemotherapy, due to their potent anti-tumor activity. The proposed research will lead to the identification and mechanistic understanding of factors that mediate interstrand cross-link repair in human cells, enabling development of more effective cancer therapeutic agents and strategies.